Electrochemical immunoassays using colloidal metal markers

ABSTRACT

The invention concerns a method for detecting or quantifying a biological substance coupled with a colloidal metal particle by electrochemical detection, characterized in that it comprises a step which consists in dissolving by chemical treatment of said colloidal metal particle, prior to detection.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional of application Ser. No. 10/311,854, filed Jun. 5,2003, now allowed, incorporated by reference herein in its entirety andrelied upon, which is a 35 U.S.C. 371 national phase application ofInternational Application No. PCT/FR01/02000, filed Jun. 25, 2001 andclaims the priority of French Application No. 00/08145 filed Jun. 26,2000.

The detection and quantification of biological substances usingimmunoassay methods or methods for assaying DNA fragments by nucleicacid hybridization are extremely important in many fields of clinicalbiology (medical and biological research, diagnosis, genetics, screeningfor illicit substances in trace amounts, etc.) or even in theenvironmental field (detection of contaminants such as pesticides orbacteria). These methods make it possible to satisfy the double criteriaof selectivity and sensitivity. Among these methods, immunoanalysis witha marker, based on antigen/antibody affinity recognition, isparticularly effective and has become widespread due to the developmentof a large range of nonradioactive markers such as enzymatic,fluorescent or chemiluminescent markers, generally coupled tospectroscopic detection. However, each marker has its own advantages anddisadvantages. Specifically, an ideal marker should satisfy variousrequirements; it should:

-   1) be detectable in a sensitive manner using analytical instruments    which are inexpensive and easy to manipulate,-   2) allow the labeled molecule (tracer) to remain soluble and stable    in the assaying media,-   3) allow simple and effective labeling at a reasonable cost,-   4) having a long lifetime,-   5) be of no risk to the individual handling it,-   6) produce a tracer having a reactivity close to the unlabeled    molecule,-   7) produce a minimal background noise.

Among the markers which have been marketed, fluorescent and luminescentmarkers, developed at the beginning of the 1970s, have many advantages:they are generally nontoxic and stable, and detection thereof is verysensitive. However, they require relatively sophisticated and expensiveequipment, and the measurement is often affected by endogenousfluorescence associated with sample matrix effects.

Enzymatic markers, which appeared at the same time as fluorescentmarkers, are probably today the most popular due to their notablecatalytic properties, but also because of their ability to trigger, forcertain substrates, colored reactions which permit the use of a verysimple detector, such as a colorimeter, or even a manipulator's eye.Enzymatic markers are the basis for methods called ELISA (Enzyme-LinkedImmunoSorbant Assay). These too have their own disadvantages however.Certain substances present in the sample may inhibit the enzyme. Inaddition, they are relatively fragile and have a limited lifetime.Moreover, the background noise can be considerable.

Metal-based markers were introduced toward the end of the 1970s, withthe aim, partly, of remedying certain of the abovementioneddisadvantages. Metal-based markers are distinguished according to theirchemical nature, namely colloidal metal particles, metal ions,coordination complexes, organometallics or else metalloproteins.Depending on their nature, various analytical techniques can beassociated with them, such as time-resolved fluorescence, atomicabsorption spectrophotometry or Fourier transform infrared, or elseelectrochemical techniques such as polarography or voltammetry.

Compared to spectrophotometric methods, electrochemical techniques havemany advantages: the measurements can be made in very small volumes ofliquid (less than a microliter), in medium which is possibly turbid (inthe case of sera), with the possibility of offering a good sensitivityfor inexpensive, possibly portable (small in size) equipment. Althoughelectrochemical techniques make it possible to detect organometallicmarkers or ionic metals down to nanomolar (10⁻⁹ M) concentrations, thisoften remains insufficient, however, compared to fluorescent markerswhich themselves can be detected down to picomolar (10⁻¹² M) thresholds.The electrochemical detection strategy developed in the presentinvention shows that it is possible to attain concentrations of ametallic marker of the order of 10⁻¹² M.

The invention relates more precisely to a method for electrochemicaldetection of a colloidal metal particle used as a marker in animmunoassay. The invention also relates to the quantitative orqualitative determination of compounds which may be haptens, antigens orantibodies, but also compounds such as DNA or RNA fragments. In general,the invention may be extended to all analytical methods involving aspecific, affinity interaction between a ligand and a host molecule, andin which it is necessary to add a marker for substantially quantifyingsaid interaction. Furthermore, many formats of immunoassays, whethercompetitive or noncompetitive, or of methods of nucleic acidhybridization, preferably in heterogeneous phase, can be applied.

The use of colloidal metal particles as a marker is not new.Specifically, they are used very commonly as a contrasting agent inelectromicroscopy techniques, in particular in the form of gold colloidscoupled to antibodies for the purpose, for example, of determining thedistribution of an antigen at the surface of a cell (Beesley,Proceedings RMS; 1985, 20, 187-196). On the other hand, the use of acolloidal metal particle as a marker in the context of an affinity assayis relatively uncommon. In this respect, the existence of a patent (U.S.Pat. No. 4,313,734) and of a publication (Leuvering et al., J.Immunoassay, 1980, 1, 71-91) relating the use of a marker based oncolloidal gold or colloidal silver in the context of an immunoassay withdetection by atomic absorption or colorimetry may be noted. Other quitesimilar patents also relate the use of gold colloid as markers inimmunoassays with colorimetric detection (U.S. Pat. No. 4,853,335,EP0310872, EP0258963). A detection, also calorimetric, has recently beendescribed for the analysis of DNA fragments by hybridization, via acolloidal gold marker (Storhoff et al., J. Am. Chem. Soc. 1998, 120,1959-1964).

As regards electrochemical detection, a method for immunoanalyzing orassaying DNA by hybridization, involving the electrochemical detectionor quantification of a colloidal metal marker, does not, for the moment,appear to have been described. The existence of an article concerningthe direct detection of a gold colloid covered with antibodies andadsorbed to the surface of a carbon paste electrode can, however, bereported (Gonzalez-Garcia and Costa-Garcia, Bioelectrochem. Bioenerg.1995, 38, 389-395). However, application to an immunoassay, althoughenvisioned, was not demonstrated.

In order to test this hypothesis, the inventors sought to verify whetheror not it was possible to carry out an immunoassay as envisioned by theauthors, i.e. an immunoassay taking place at the very surface of theelectrode and for which, after immunoreaction, the colloidal gold markerwhich has reacted in the proximity of the surface of the electrode isdirectly detected. The result of this experiment made it possible toconclude that it was not possible to detect the gold colloid in thisway, probably because the latter is no longer in immediate contact withthe surface of the electrode. The present invention makes it possible tobe free of this problem by virtue of an indirect detection of thecolloidal metal marker, which by the same token allows the use of asolid phase which may be different from the surface of the electrode.

Thus, the present invention allows the detection or quantification of abiological substance coupled to a colloidal metal particle, byelectrochemical detection, said colloidal metal particle beingdissolved, and detected by electrochemistry after having beenreprecipitated at the surface of the electrode. This makes it possibleto increase the local concentration and the sensitivity threshold. Thesubject of the invention is therefore a method for detection orquantification of a biological substance coupled to a colloidal metalparticle, by electrochemical detection, characterized in that itcomprises a step of dissolving said colloidal metal particle.

The electrochemical immunoassay method developed in the presentinvention can not only be more sensitive than the current enzymaticimmunoassay techniques, but also offer the possibility of determiningand/or quantifying several compounds simultaneously if several colloidalmetal markers which are different in nature are used. Specifically,electrochemical methods permit the simultaneous detection of severalmetals in the course of the same measurement. In addition, colloidalmetal markers offer the advantage of being much more stable thanradioisotopic or enzymatic markers, and they permit simple labeling ofmany substances, at low cost, without any loss of activity of thesesubstances.

The chemical treatment to dissolve the colloidal metal particle iscarried out in an acidic medium containing an oxidant. The concentrationof oxidant is chosen so as to be in sufficient excess to dissolve thehighest concentrations of colloidal metal marker. A solution ofhydrobromic acid containing bromine (Br₂) or hypobromous acid (HBrO) ora mixture of the two as oxidant is preferred (for example: 10⁻⁴ M of Br₂in 0.1 or 1 M HBr), in particular when dissolution of a gold colloid isdesired. A solution of hydrochloric acid (for example 0.1 M) containinga bromide salt (concentration ≧0.1 M) and bromine may also be suitable.Depending on the nature of the metal colloid to be dissolved, otheracidic media for dissolution (H₂SO₄, HClO₄, HF, etc.) and oxidizingreagents (I₂, Cl₂, HClO, HIO, H₂O₂, HNO₃, CN⁻, Cr₂O₄ ⁻, MnO₄ ⁻, . . . )may be envisioned.

After dissolution, an additional treatment may be necessary to removethe excess oxidizing reagent such as bromine. To do this, an excess ofphenol, aniline, hydrazine, oxine or one of their derivatives, or elsepreferably an excess of 3-phenoxyacetic acid, may be added to themedium. The latter is preferable since it is less toxic. A concentrationof 5×10⁻⁴ M is generally sufficient. The bromine may also be removed bydegassing.

It may be advantageous to add in solution a reagent capable ofcomplexing the metal ion in order to promote detection thereof. Indeed,complexation can transform a nonelectroactive metal ion into adetectable electroactive compound. In addition, the complexed metal ion,because it has a more marked hydrophobic nature, can adsorb to theelectrode and thus be more detected with more sensitively by adsorptivecathodic stripping voltammetry (van den Berg, Anal. Chim. Acta, 1991,250, 265-276).

After the metal has dissolved, it is reduced at the surface of theelectrode, preferably by applying a very negative potential. Thepotential is then varied in order to reoxidize the metal, which thengoes into solution. The intensity of the voltammetric peak (surface)reflects the amount of metal deposited on the electrode, and thereforethe amount of colloidal particles initially present in the solution.This therefore makes it possible to perform the assaying. When particlesconsisting of different metals are used, simultaneous detection ispossible due to the distinct reoxidation potentials of the variousmetals.

Thus, it is possible to deduce the presence and/or the amount ofbiological substance initially coupled to the colloidal particle, as afunction of the presence and/or amount of metal electrodeposited at thesurface of the electrode.

The colloidal metal particles may consist of metal, such as gold,silver, copper, platinum, rhodium, palladium, iridium, nickel or ironcolloids, or else of metal compounds, such as, for example, metal oxidesor halides or chalcogenides, such as Ag₂O, AgI, Bi₂O₅, Cd₃P₂, CdS, CdSe,CdTe, Co₂O₃, CrO₃, Cu₂S, HgI₂, MnO₂, PbS, PbO₂, SnO₂, TiO₂, RuO₂, ZnO,ZnS or ZnO₂, or metal hydroxides. In general, any metal or metalcompound which can be detected electrochemically can be envisioned,preferably transition metals (van den Berg, Anal. Chim. Acta, 1991, 250,265-276). Practical requirements mean that preference is given to theuse of metals or metal compounds which are only barely present, or notat all, in the assaying medium, and more particularly those which offerthe best detection limits with respect to the electrochemical techniquesused. The invention has been demonstrated in particular for a goldcolloid.

The metal-based colloidal particles can be obtained using one of themany methods described in the scientific literature (Hayat, Ed,Colloidal gold: principles, methods, and applications; Academic Press:San Diego, Calif., 1991—Mackay and Texter, Eds, Electrochemistry incolloids and dispersions, VCH Publishers: New York, 1992—Murray et al.,J. Am. Chem. Soc. 1993, 115, 8706-8715—Frens, Nat. Phys. Sci. 1973, 241,20-22—U.S. Pat. No. 5,637,508—Weller, Angew. Chem. Int. Ed. Engl. 1993,32, 41-43—Wang and Herron, J. Phys. Chem. 1991, 95, 525-532). Dependingon the method of production, the particles may be between 1 and 200 nmin size, with a very low dispersion. In the present invention, metalcolloids between 5 and 100 nm are preferred. The use of a particle ofconsiderable size is liable to improve the sensitivity of the assay.

Depending on the format and the type of assay, the colloidal metalparticle can be coupled to an antibody, a protein receptor, an antigen,a hapten, a protein, a peptide, an oligonucleotide or a nucleic acidfragment (in particular DNA or RNA). The term “coupling” is intended tomean any method of chemical or physical attachment, direct or indirect,to the surface of the particle, such as a covalent bond or an adsorptionvia electrostatic interactions, hydrogen bridges, etc. Many couplingprotocols have been described (Beesley, Proceedings RMS, 1985, 20,187-196—Oliver, Methods in molecular biology, 1999, 115, 331-334). Thespecies then labeled with the metal particle is then used as a reagentwhich, in combination with immunochemical reagents based on antibodies,protein receptors, haptens, antigens, proteins, peptides,oligonucleotides, or DNA or RNA fragments, buffer solutions, otherchemical reagents, and an electrode-based electrochemical detectionsystem, will make it possible to assay a given substance.

The principle of the invention is, by way of example, illustrated inFIG. 1, in the case of a sandwich-type noncompetitive immunoassay (FIG.1A), and also for a competitive immunoassay (FIG. 1B).

For the first approach (FIG. 1A), the compound to be determined (theanalyte) is initially captured with a first ligand (in the present casean antibody; for a hybridization assay, this would be anoligonucleotide) immobilized on a solid phase. The solid phase forimmobilizing the ligand may, for example, be the bottom of amicrocuvette (in the case of FIG. 1), the surface of a microbead(optionally magnetic), the surface of a membrane or else the surface ofthe electrode. After a given incubation period, optionally followed by awashing step, a second ligand (here an antibody) labeled with a metalcolloid is added in such a way that it reacts with the analytepreviously extracted on the solid phase. The solid phase thusconstituted is then washed then treated with an appropriate volume of asolution of reagent able to dissolve the colloidal metal marker whichhas reacted with the solid phase. Then, the metal thus solubilized inthe ionic state is detected and quantified using an electrode eitherimmersed in the solution (in situ method—FIG. 1A), or after transfer ofthe solution (ex situ method). The electrochemical response can then bequalitatively or quantitatively linked to the substance to be assayed.

In the case of the second approach (FIG. 1B), the method consists inbringing a sample containing the substance to be assayed into contactwith a known amount of said substance labeled with a metal colloid, anda certain amount of ligand (antibody) immobilized on a solid phase anddirected against this substance. After a given reaction time, the natureand the amount of metal colloid present in the fraction bound is then,after an optional washing step, determined as indicated above afterdissolution and then electrochemical detection.

The use of a solid phase consisting of microbeads, for example made oflatex or else of ferromagnetic oxide, may be particularly advantageousfor improving the sensitivity and lowering the limit of detection.Specifically, the microbeads can be concentrated, after theimmunoreaction step, on a small surface, such as, for example, thesurface of a filtering membrane (U.S. Pat. No. 4,853,335—Tu et al.,Anal. Chem. 1993, 65, 3631-3665) or else the bottom of a conical tube,thus offering the possibility of dissolving the metal colloid in asmaller volume of liquid than that in which the immunoreaction tookplace.

Methods based on agglutination and/or precipitation in homogeneous phaseof the colloidal metal marker in the course of an immunoreaction oroligonucleotide hybridization can also be envisioned (U.S. Pat. No.5,851,777). In this case, the aggregates formed are isolated and thendissolved and detected as previously.

As regards the nature of the electrodes, carbon-based electrodes arepreferred, and more particularly disk electrodes (FIG. 2A) and stripmicroelectrodes (FIG. 2B) obtained by screen-printing with acarbon-based ink. These electrodes are in fact particularly well suitedsince they can be mass-produced at low cost, and can thereforeoptionally be for single use. In addition, their geometric form and alsotheir size can be readily modifiable. However, other types of electrodemay be used, such as electrodes made of glass carbon, graphite,composite materials containing carbon, carbon fibers, and/or carbonpaste. In addition, the surface of the electrodes can be treatedelectrochemically or chemically in order to improve the sensitivity ofdetection of the dissolved metal (Kalcher, Electroanalysis, 1990, 2,419-433—Kalcher et al., Electroanalysis, 1995, 7, 5-22—Ugo and Moretto,Electroanalysis, 1995, 7, 1105-1113). This may be, by way of example,modification of the surface of the electrode or of the composition ofthe ink, with a polymer capable of attracting metal ions viaelectrostatic interaction or complexation, or else electrochemicalpretreatment of the surface of the electrode. Depositing a mercury filmmay also prove to be advantageous for certain metal ions which aredifficult to detect on a carbon electrode. As regards the method ofproducing the electrodes, the screen-printing technique is preferable,although other methods of industrial production, such as rotogravure,inkjet printing, or optionally photolithography may be adapted.

According to the characteristics of the invention, the use ofmicroelectrodes, preferably of strip microelectrodes, obtained byscreen-printing is particularly advantageous since they offer thepossibility of carrying out measurements in very small volumes (of theorder of a few microliters) for analytical performances which, in termsof sensitivity and limit of detection of metal ion, are generallyimproved (Wong and Ewing, Anal. Chem. 1990, 62, 2697-2702—Wang et al.,J. Electroanal. Chem., 1993, 361, 77-83—Wang and Armalis,Electroanalysis, 1995, 7, 958-961—Alames-Varela and Costa-Garcia,Electroanalysis, 1997, 9, 1262-1266).

FIG. 3, which compares the calibration curves (current densities) ofAuBr₄ ⁻ in 0.1 M HBr, obtained on a screen-printed strip microelectrodewith a surface area S=1.7×10⁻⁴ cm² (curve 1) and on a screen-printeddisk macroelectrode with a surface area S=0.0962 cm² (curve 2), confirmsbetter sensitivity in the case of the strip microelectrode. These curveswere obtained by linear anodic stripping voltammetry in the followingway: (I) electrodeposition of gold at a constant potential of E=−0.3 Vfor 300 s, (ii) then linear potential scan from 0.2 V up to 1.1 V at arate of 50 mVs⁻¹. The peak current (i_(p)) appears around 1.0 V, linkedto oxidation of the gold, and is taken as the analytical response.

It in fact appears that the use of microelectrodes makes it possible toobtain deposition of the metal which is more effective than with amacroelectrode. Specifically, the use of macroelectrodes requires thesolution to be agitated in order to ensure that a sufficient amount ofmetal deposits at the surface of the electrode. Surprisingly, theinventors have observed that the use of microelectrodes makes itpossible to do without this agitation step. This may explain the gain insensitivity observed, although other hypotheses, due to the very natureof the microelectrode (very small in size), may also be envisioned.

Various techniques of electrochemical analysis may be used to assay thedissolved metal ions. They are preferentially anodic strippingvoltammetry (or polarography) with a potential scan which may be linear,cyclic, square-ware, normal pulse or differential pulse, or with asuperimposed sinusoidal voltage, or else anodic strippingchronopotentiometry. However, other techniques may be used, such as ionexchange voltammetry, adsorptive cathodic stripping voltammetry (orpolarography) with a scan which may be linear, cyclic, square-ware,normal pulse or differential pulse, or with a superimposed sinusoidalvoltage, or else chronoamperometry, chronocoulometry or linear, cyclic,square-wave, normal pulse or differential pulse voltammetry (orpolarography) or voltammetry (or polarography) with a superimposedsinusoidal voltage. These techniques require a possibly two-electrode oreven three-electrode assembly, i.e. an assembly comprising theabovementioned measuring electrode, a reference electrode and,optionally, an auxiliary electrode. In order to avoid contamination ofthe assaying medium with the metal or the electrolyte of the referenceelectrode, it is advantageous to isolate this electrode with anextension which consists, at its end, of a porous material, and which isfilled with an electrolyte. A reference electrode screen-printed usingan ink based on silver and silver chloride may also be envisioned. Hereagain, it may be useful to isolate this electrode via an electrolytebridge, such as, for example, an ionic conducting gel or an ionicconducting polymer, in order to avoid interference from the silver ionsduring a measurement.

The invention also relates to a kit for assaying at least one biologicalcompound. According to the characteristics of the invention, this kitcomprises at least one reagent labeled with a colloidal metal particleand at least one electrode. The kit according to the invention may alsocontain at least one reagent for dissolving the colloidal metal particleand, optionally, a reagent for eliminating the excess oxidizing reagent.The kit may also contain a reagent capable of complexing the metal ion,in order to promote detection thereof. Finally, the kit may also containinstructions in order to enable the method according to the presentinvention to be carried out.

The following examples illustrate the characteristics of the invention,but should not be considered to limit the invention.

DESCRIPTION OF THE FIGURES

FIG. 1. Diagrammatic representation of the principle of the inventionillustrated in the case (A) of a noncompetitive immunoassay and (B) acompetitive immunoassay.

FIG. 2. Diagrammatic representation of a screen-printed disk electrode(A) and microstrip electrode (B).

FIG. 3. Calibration curves for ionic gold, obtained (1) with a stripmicroelectrode and (2) with a disk electrode.

FIG. 4. Diagrammatic representation of the measuring device used todetect the gold dissolved in a small volume of a drop of solution.

FIG. 5. Calibration curves for two colloids of gold covered withstreptavidin.

FIG. 6. (A) log-log calibration curve for the IgG noncompetitiveimmunoassay. (B) anodic stripping voltammetry curves obtained forvarious concentrations of IgG. The curves are identified by letters inorder to make them correspond to the concentrations indicated by thesame letters on the IgG calibration curve.

FIG. 7. Log-log calibration curve for the α-feto-protein noncompetitiveimmunoassay.

EXAMPLES Example 1 Detection of Streptavidin Labeled with a Gold Colloidafter Specific Attachment to the Bottom of a Microwell

The experiments are carried out at ambient temperature.

The bovine serum albumin (BSA, fraction V), the biotin-amidocaproylcoupled to BSA (B-BSA, biotin content: 8-12 mol/mol of BSA), thestreptavidin labeled with colloidal gold (S—Au) 20 nm in diameter, andalso the streptavidin coupled to albumin onto which 10 nm colloidal goldparticles are adsorbed (SA-Au) come from Sigma Chemical Co.

100 μl of B-BSA at 10 μg ml⁻¹ in a bicarbonate buffer (15 mM Na₂CO₃; pH9.6) are pipetted at the bottom of a polystyrene microwell (Nunc) anallowed to incubate for 2 hours. After having emptied the microwell andrinsed it with 110 μl of phosphate buffer (PBS: 4.3 mM NaH₂PO₄, 15.1 mMNa₂HPO₄ and 50 mM NaCl; pH 7.4), 100 μl of PBS containing 0.1% of BSA(PBS-BSA) are added and allowed to incubate for 2 hours. The microwellis then emptied and rinsed 3 times with 110 μl of pure water. Next, 35μl of a solution of S—Au or SA-Au at x μg.ml⁻¹ (0.003<x<3) in a PBS-BSAbuffer containing 0.05% of Tween 20 (PBS-BSA-T) are then introduced intothe microwell and allowed to react for 3 hours. Once emptied, themicrowell is thoroughly washed with 3×110 μl of PBS-BSA-T, and then with2×110 μl of PBS. The gold colloid attached to the walls of the microwellis then dissolved by introducing 40 μl of a solution of Br₂ at aconcentration of 10⁻⁴ M in 1 M HBr. After 5 minutes, a volume of 35 μlis removed from the microwell and transferred onto the surface of ascreen-printed carbon disk electrode (S=0.0962 cm², electrode preparedaccording to the method described in the ref.: Bagel et al., Anal. Chem.1997, 69, 4688-4694), to which are added 5 μl of a fresh solution of3-phenoxypropionic acid at 4×10⁻³ M in 1 M HBr. A reference electrode(Ag/AgBr, NaBr_(set)) extended via an extension containing a saturatedsolution of NaBr, and an auxiliary electrode are them immersed in the 40μl of solution previously deposited onto the surface of thescreen-printed carbon electrode, as shown by the diagram in FIG. 4. Thelinear anodic stripping voltammetry measurements are then carried out inthe following way:

-   1) electrodeposition of the gold at a constant potential of E=−0.3 V    for 300 s,-   2) then a linear potential scan from 0.2 V up to 1.1 V at a rate of    50 mVs⁻¹.

The peak current (i_(p)) which appears around 1.0 V, linked to oxidationof the gold, is taken as the analytical response. The measurement mayalso be the integral of the peak, which then corresponds to a coulombquantity (Q_(p)). The calibration curves are represented in FIG. 5, on alogarithmic scale, for each of the streptavidins labeled with gold.Better sensitivity with the SA-Au (curve 1) than with the S—Au (curve 2)can be noted.

Example 2 “Sandwich” Immunoassay for an Immunoglobulin

The ovalbumin (OA, grade III) and the goat immunoglobulin G (IgG) ismarketed by Sigma Chemical Co. The anti-goat IgG labeled with an 18 nmgold colloid, and also the unlabeled anti-goat IgG, are polyclonalantibodies from the Jackson Immunoresearch Laboratories.

60 μl of a solution of anti-IgG at 24 μg ml⁻¹ in a PBS buffer arepipetted into a microwell and allowed to incubate for 1 hour. Afterhaving emptied the microwell and rinsed it with 2×100 μl of PBS buffercontaining 0.5% of ovalbumin and 0.1% of Tween 20 (PBS-OA-T), 100 μl ofthis same buffer are then added and allowed to incubate for 1 hour. Thesolution is then drawn off, and 35 μl of a solution of goat IgG at xng.ml⁻¹ (0.5<x<1 000) diluted in a PBS buffer containing 0.1% of Tween20 are then introduced and allowed to incubate for 40 minutes. Once themicrowell has been emptied and rinsed with 2×100 μl of PBS-OA-T 100 μlof PBS-OA-T are introduced. After 30 minutes, the liquid is replacedwith 35 μl of a dilute solution of anti-IgG labeled with colloidal gold(45-fold dilution of the solution marketed, in PBS-OA-T), and thenincubated for 3 hours. A final rinsing cycle is carried out, washing themicrowell 3 times with 200 μl of PBS-OA-T, followed by 2×100 μl of PBS.The liquid is then carefully drawn off, and the gold colloid attached tothe walls of the microwell is then dissolved and detected as indicatedin example 1.

Some examples of measurements obtained by linear anodic strippingvoltammetry are given in FIG. 6A, while the corresponding goat-IgGcalibration curve is represented in FIG. 6B. Each point represents themean of 2 measurements and each measurement was obtained with adifferent electrode (single-use electrode). A concentration ofapproximately 3×10⁻¹² M of IgG could be determined.

Example 3 Noncompetitive Immunoassay for Human α-feto-protein

80 μl of a solution of monoclonal anti-α-fetoprotein (mouse antibody) at24 μg ml⁻¹ in a carbonate buffer (15 mM, pH 9.6) are pipetted into amicrowell and allowed to incubate overnight at 4° C. After havingemptied the microwell and rinsed it with 2×250 μl of PBS-OA-T buffer,250 μl of this same buffer are then added and allowed to incubate for 40min. The solution is then drawn off, and 80 μl of a solution ofα-fetoprotein at x ng.ml⁻¹ (0.05<x<20) diluted in a PBS buffercontaining 0.1% of Tween 20 are then introduced and allowed to incubatefor 2 hours. Once the microwell has been emptied and rinsed with 2×250μl of PBS-OA-T, 250 μl of PBS-OA-T are introduced. After 30 minutes, theliquid is replaced with 80 μl of a dilute solution of polyclonalanti-α-fetoprotein (goat antibody) diluted to 5 μl ml⁻¹ in PBS-OA-T, andincubated for 1 hour. Then, after rinsing with 2×250 μl of PBS-OA-Tbuffer, 50 μl of a solution of anti-goat IgG labeled with colloidal gold(45-fold dilution of the solution marketed, in PBS-OA-T) [lacuna], andthen incubated for 1 h 30 min. A final rinsing cycle is carried out,washing the microwell 3 times with 250 μl of PBS-OA-T, followed by 2×250μl of PBS-T, then 2×250 μl of PBS. The liquid is then carefully drawnoff, then the gold colloid attached to the walls of the microwell isthen dissolved and detected with strip microelectrodes in the followingway: 50 μl of a solution of 0.1 mM Br₂ in 0.1 M HBr are added to themicrowells for 30 min; then 40 μl are transferred into new microwellscontaining 10 μl of a solution of 3-phenoxypropionic acid at 2×10⁻³ M in0.1 N HBr. Detection of the dissolved gold is then carried out asindicated in example 1.

Production of Screen-Printed Strip Microelectrodes:

The carbon-based ink used to produce the strip micro-electrodes is acommercial ink produced by Acheson Colloid (Minico® inks of theM3000-1RS series or Electrodag® inks such as 423 SS or PF 407A). The inkis screen-printed onto a rigid or semi-rigid support, preferably made ofsemicrystalline polystyrene or high-impact polystyrene (plates possiblybetween 0.1 and 2 mm thick). The fineness of the screen-printing maskobtained on a taut screen mounted on a frame, and also the nature andthe mesh size of the screen, condition to a large extent the quality ofthe ink deposit and also its thickness. In the present invention, inkthicknesses of between 5 and 50 μm could be obtained usingscreen-printing frames comprising 77 or 120 threads/cm.

Once the ink has been screen-printed, it is left to dry in an oven atbetween 60 and 100° C. Next, a polystyrene-based isolating layer isdeposited or screen-printed in such a way as to re-cover part of thecarbon ink previously screen-printed (FIG. 2). After drying, theelectrode thus constituted is cut up transversely along its thickness soas to reveal on the section a strip of carbon of micrometric thickness(depending on the thickness of carbon ink initially screen-printed) andof millimetric length (depending on the length of the motif of theelectrode initially selected) (FIG. 2B).

1. A diagnostic kit, comprising at least one reagent labeled withcolloidal metal particles and at least one electrode, and also an agentfor chemically dissolving said colloidal metal particles.
 2. The kitaccording to claim 1, further comprising a reagent capable of complexingthe metal ions.
 3. The kit according to claim 1, further includinginstructions for the electrochemical detection or quantification of abiological substance using said kit.
 4. The kit according to claim 1,wherein the agent for dissolving said colloidal metal particles is anacidic medium containing an oxidant.
 5. The kit according to claim 2,wherein the reagent capable of complexing the metal ions is in solution.6. The kit as claimed in claim 1, wherein the colloidal particles areselected from the group consisting of particles comprising metal andmetal compounds.
 7. The kit as claimed in claim 6, wherein the colloidalparticles are gold.
 8. The kit as claimed in claim 1, wherein thecolloidal particles are each between 1 and 200 nm in size.
 9. The kit asclaimed in claim 4, wherein the acidic medium containing an oxidant is asolution of hydrobromic acid containing bromine, hypobromous acid or amixture of these two compounds.
 10. The kit as claimed in claim 1,wherein the electrode is a screen-printed electrode.